RESUMO
The budding yeast Mre11-Rad50-Xrs2 (MRX) complex and Sae2 function together in DNA end resection during homologous recombination. Here we show that the Ku complex shields DNA ends from exonucleolytic digestion but facilitates endonucleolytic scission by MRX with a dependence on ATP and Sae2. The incision site is enlarged into a DNA gap via the exonuclease activity of MRX, which is stimulated by Sae2 without ATP being present. RPA renders a partially resected or palindromic DNA structure susceptible to MRX-Sae2, and internal protein blocks also trigger DNA cleavage. We present models for how MRX-Sae2 creates entry sites for the long-range resection machinery.
Assuntos
Reparo do DNA por Junção de Extremidades , Reparo do DNA/fisiologia , Endonucleases/metabolismo , Exonucleases/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Clivagem do DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Ativação Enzimática/genética , Exodesoxirribonucleases/metabolismo , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
DNA double-strand break repair by homologous recombination entails nucleolytic resection of the 5' strand at break ends. Dna2, a flap endonuclease with 5'-3' helicase activity, is involved in the resection process. The Dna2 helicase activity has been implicated in Okazaki fragment processing during DNA replication but is thought to be dispensable for DNA end resection. Unexpectedly, we found a requirement for the helicase function of Dna2 in end resection in budding yeast cells lacking exonuclease 1. Biochemical analysis reveals that ATP hydrolysis-fueled translocation of Dna2 on ssDNA facilitates 5' flap cleavage near a single-strand-double strand junction while attenuating 3' flap incision. Accordingly, the ATP hydrolysis-defective dna2-K1080E mutant is less able to generate long products in a reconstituted resection system. Our study thus reveals a previously unrecognized role of the Dna2 translocase activity in DNA break end resection and in the imposition of the 5' strand specificity of end resection.
Assuntos
DNA Helicases/metabolismo , Reparo do DNA/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Região 5'-Flanqueadora/genética , Trifosfato de Adenosina/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , DNA Helicases/genética , Mutação , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Nucleolytic resection of DNA double-strand breaks is the crucial first step in their repair via homologous recombination. New findings by Tkác et al. (2016) published in this issue of Molecular Cell identify HELB as a novel, cell-cycle-specific negative regulator of DNA end resection.
Assuntos
Reparo do DNA por Junção de Extremidades , DNA Helicases/metabolismo , Neoplasias Mamárias Experimentais/enzimologia , Animais , Feminino , HumanosRESUMO
The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5'-ended DNA strands at DSB ends, producing 3'-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Peptídeos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Sequência de Aminoácidos , Caseína Quinase II/metabolismo , Sequência Conservada , Quebras de DNA de Cadeia Dupla , FosforilaçãoRESUMO
The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand-displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair.
Assuntos
DNA Helicases , Reparo de Erro de Pareamento de DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo de Erro de Pareamento de DNA/genética , DNA Polimerase III/metabolismo , HumanosRESUMO
New findings on the RIF1 protein by four research groups, including Chapman et al. (2013) and Escribano-Díaz et al. (2013) in this issue, provide insights into DNA double-strand break repair pathway choice in mammalian cells.
RESUMO
Bloom helicase (BLM) and its orthologs are essential for the maintenance of genome integrity. BLM defects represent the underlying cause of Bloom Syndrome, a rare genetic disorder that is marked by strong cancer predisposition. BLM deficient cells accumulate extensive chromosomal aberrations stemming from dysfunctions in homologous recombination (HR). BLM participates in several HR stages and helps dismantle potentially harmful HR intermediates. However, much remains to be learned about the molecular mechanisms of these BLM-mediated regulatory effects. Here, we use DNA curtains to directly visualize the activity of BLM helicase on single molecules of DNA. Our data show that BLM is a robust helicase capable of rapidly (â¼70-80 base pairs per second) unwinding extensive tracts (â¼8-10 kilobases) of double-stranded DNA (dsDNA). Importantly, we find no evidence for BLM activity on single-stranded DNA (ssDNA) that is bound by replication protein A (RPA). Likewise, our results show that BLM can neither associate with nor translocate on ssDNA that is bound by the recombinase protein RAD51. Moreover, our data reveal that the presence of RAD51 also blocks BLM translocation on dsDNA substrates. We discuss our findings within the context of potential regulator roles for BLM helicase during DNA replication and repair.
Assuntos
DNA de Cadeia Simples/metabolismo , DNA/metabolismo , RecQ Helicases/análise , RecQ Helicases/metabolismo , Imagem Individual de Molécula , Pareamento de Bases , Síndrome de Bloom/genética , DNA/química , Reparo do DNA/genética , Replicação do DNA/genética , DNA de Cadeia Simples/química , Recombinação Homóloga , Humanos , Modelos Moleculares , Rad51 Recombinase/metabolismo , RecQ Helicases/química , RecQ Helicases/genética , Proteína de Replicação A/metabolismo , Imagem Individual de Molécula/métodosRESUMO
The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic resection of the DNA break ends. The current model, being based primarily on genetic analyses in Saccharomyces cerevisiae and companion biochemical reconstitution studies, posits that end resection proceeds in two distinct stages. Specifically, the initiation of resection is mediated by the nuclease activity of the Mre11-Rad50-Xrs2 (MRX) complex in conjunction with its cofactor Sae2, and long-range resection is carried out by exonuclease 1 (Exo1) or the Sgs1-Top3-Rmi1-Dna2 ensemble. Using fully reconstituted systems, we show here that DNA with ends occluded by the DNA end-joining factor Ku70-Ku80 becomes a suitable substrate for long-range 5'-3' resection when a nick is introduced at a locale proximal to one of the Ku-bound DNA ends. We also show that Sgs1 can unwind duplex DNA harboring a nick, in a manner dependent on a species-specific interaction with the ssDNA-binding factor replication protein A (RPA). These biochemical systems and results will be valuable for guiding future endeavors directed at delineating the mechanistic intricacy of DNA end resection in eukaryotes.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , RecQ Helicases/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , DNA Helicases/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases/genética , Recombinação Homóloga , RecQ Helicases/genética , Proteína de Replicação A/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
DNA double-strand breaks can be eliminated via non-homologous end joining or homologous recombination. Non-homologous end joining is initiated by the association of Ku with DNA ends. In contrast, homologous recombination entails nucleolytic resection of the 5'-strands, forming 3'-ssDNA tails that become coated with replication protein A (RPA). Ku restricts end access by the resection nuclease Exo1. It is unclear how partial resection might affect Ku engagement and Exo1 restriction. Here, we addressed these questions in a reconstituted system with yeast proteins. With blunt-ended DNA, Ku protected against Exo1 in a manner that required its DNA end-binding activity. Despite binding poorly to ssDNA, Ku could nonetheless engage a 5'-recessed DNA end with a 40-nucleotide (nt) ssDNA overhang, where it localized to the ssDNA-dsDNA junction and efficiently blocked resection by Exo1. Interestingly, RPA could exclude Ku from a partially resected structure with a 22-nt ssDNA tail and thus restored processing by Exo1. However, at a 40-nt tail, Ku remained stably associated at the ssDNA-dsDNA junction, and RPA simultaneously engaged the ssDNA region. We discuss a model in which the dynamic equilibrium between Ku and RPA binding to a partially resected DNA end influences the timing and efficiency of the resection process.
Assuntos
Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Recombinação Homóloga/genética , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases/genética , Regulação Fúngica da Expressão Gênica , Proteína de Replicação A/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
BLM, a RecQ family DNA helicase mutated in Bloom's Syndrome, participates in homologous recombination at two stages: 5' DNA end resection and double Holliday junction dissolution. BLM exists in a complex with Topo IIIα, RMI1 and RMI2. Herein, we address the role of Topo IIIα and RMI1-RMI2 in resection using a reconstituted system with purified human proteins. We show that Topo IIIα stimulates DNA unwinding by BLM in a manner that is potentiated by RMI1-RMI2, and that the processivity of resection is reliant on the Topo IIIα-RMI1-RMI2 complex. Topo IIIα localizes to the ends of double-strand breaks, thus implicating it in the recruitment of resection factors. While the single-stranded DNA binding protein RPA plays a major role in imposing the 5' to 3' polarity of resection, Topo IIIα also makes a contribution in this regard. Moreover, we show that DNA2 stimulates the helicase activity of BLM. Our results thus uncover a multifaceted role of the Topo IIIα-RMI1-RMI2 ensemble and of DNA2 in the DNA resection reaction.
Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Reparo do DNA , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , RecQ Helicases/metabolismo , Proteínas de Transporte/fisiologia , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Helicases/fisiologia , DNA Topoisomerases Tipo I/fisiologia , Proteínas de Ligação a DNA/fisiologia , Endodesoxirribonucleases/metabolismo , Humanos , Proteínas Nucleares/fisiologiaRESUMO
Homologous recombination (HR) is an evolutionarily conserved process that eliminates DNA double-strand breaks from chromosomes, repairs injured DNA replication forks, and helps orchestrate meiotic chromosome segregation. Recent studies have shown that DNA helicases play multifaceted roles in HR mediation and regulation. In particular, the S. cerevisiae Sgs1 helicase and its human ortholog BLM helicase are involved in not only the resection of the primary lesion to generate single-stranded DNA to prompt the assembly of the HR machinery, but they also function in somatic cells to suppress the formation of chromosome arm crossovers during HR. On the other hand, the S. cerevisiae Mph1 and Srs2 helicases, and their respective functional equivalents in other eukaryotes, suppress spurious HR events and favor the formation of noncrossovers via distinct mechanisms. Thus, the functional integrity of the HR process and HR outcomes are dependent upon these helicase enzymes. Since mutations in some of these helicases lead to cancer predisposition in humans and mice, studies on them have clear relevance to human health and disease.
Assuntos
DNA Helicases , Saccharomyces cerevisiae , Animais , DNA Helicases/metabolismo , Reparo do DNA , Recombinação Homóloga , Humanos , Negociação , RecQ Helicases/genética , Recombinação Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The DNA double-strand break (DSB), arising from exposure to ionizing radiation or various chemotherapeutic agents or from replication fork collapse, is among the most dangerous of chromosomal lesions. DSBs are highly cytotoxic and can lead to translocations, deletions, duplications, or mutations if mishandled. DSBs are eliminated by either homologous recombination (HR), which uses a homologous template to guide accurate repair, or by nonhomologous end joining (NHEJ), which simply rejoins the two broken ends after damaged nucleotides have been removed. HR generates error-free repair products and is also required for generating chromosome arm crossovers between homologous chromosomes in meiotic cells. The HR reaction includes several distinct steps: resection of DNA ends, homologous DNA pairing, DNA synthesis, and processing of HR intermediates. Each occurs in a highly regulated fashion utilizing multiple protein factors. These steps are being elucidated using a combination of genetic tools, cell-based assays, and in vitro reconstitution with highly purified HR proteins. In this review, we summarize contributions from our laboratory at Yale University in understanding HR mechanisms in eukaryotic cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA Cruciforme/genética , Recombinação Homóloga , Transdução de Sinais/genética , DNA Cruciforme/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Genéticos , Rad51 Recombinase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Apurinic/apyrimidinic (AP) endonucleases are versatile DNA repair enzymes that possess a variety of nucleolytic activities, including endonuclease activity at AP sites, 3' phosphodiesterase activity that can remove a variety of ligation-blocking lesions from the 3' end of DNA, endonuclease activity on oxidative DNA lesions, and 3' to 5' exonuclease activity. There are two families of AP endonucleases, named for the bacterial counterparts endonuclease IV (EndoIV) and exonuclease III (ExoIII). While ExoIII family members are present in all kingdoms of life, EndoIV members exist in lower organisms but are curiously absent in plants, mammals and some other vertebrates. Here, we review recent research on these enzymes, focusing primarily on the EndoIV family. We address the role(s) of EndoIV members in DNA repair and discuss recent findings from each model organism in which the enzymes have been studied to date.
Assuntos
Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Desoxirribonuclease IV (Fago T4-Induzido)/genética , Sequência de Aminoácidos , Animais , Pareamento Incorreto de Bases , Caenorhabditis elegans , DNA Fúngico/genética , DNA Mitocondrial/genética , Humanos , Dados de Sequência Molecular , Nucleotídeos/genética , Schizosaccharomyces/genética , Homologia de Sequência de Aminoácidos , Xenopus/genética , Peixe-ZebraRESUMO
DNA double-strand break repair by homologous recombination begins with nucleolytic resection of the 5' DNA strand at the break ends. Long-range resection is catalyzed by EXO1 and BLM-DNA2, which likely have to navigate through ribonucleotides and damaged bases. Here, we show that a short stretch of ribonucleotides at the 5' terminus stimulates resection by EXO1. Ribonucleotides within a 5' flap are resistant to cleavage by DNA2, and extended RNA:DNA hybrids inhibit both strand separation by BLM and resection by EXO1. Moreover, 8-oxo-guanine impedes EXO1 but enhances resection by BLM-DNA2, and an apurinic/apyrimidinic site stimulates resection by BLM-DNA2 and DNA strand unwinding by BLM. Accordingly, depletion of OGG1 or APE1 leads to greater dependence of DNA resection on DNA2. Importantly, RNase H2A deficiency impairs resection overall, which we attribute to the accumulation of long RNA:DNA hybrids at DNA ends. Our results help explain why eukaryotic cells possess multiple resection nucleases.
Assuntos
Quebras de DNA de Cadeia Dupla , Ribonucleotídeos/genética , Ribonucleotídeos/metabolismo , Western Blotting , Linhagem Celular Tumoral , DNA Glicosilases/genética , Enzimas Reparadoras do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Exodesoxirribonucleases/genética , Imunofluorescência , Recombinação Homóloga/genética , Humanos , RecQ Helicases/genética , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Nonhomologous end joining (NHEJ) directly rejoins DNA double-strand breaks (DSBs) when recombination is not possible. In Saccharomyces cerevisiae, the DNA polymerase Pol4 is required for gap filling when a short 3' overhang must prime DNA synthesis. Here, we examined further end variations to test specific hypotheses regarding Pol4 usage in NHEJ in vivo. Surprisingly, Pol4 dependence at 3' overhangs was reduced when a nonhomologous 5' flap nucleotide was present across from the gap, even though the mismatched nucleotide was corrected, not incorporated. In contrast, a gap with a 5' deoxyribosephosphate (dRP) was as Pol4-dependent as a gap with a 5' phosphate, demonstrating the importance of the downstream base in relaxing the Pol4 requirement. Combined with prior observations of Pol4-independent NHEJ of nicks with 5' hydroxyls, we suggest that base stacking interactions across the broken strands can stabilize a joint, allowing another polymerase to substitute for Pol4. This model predicts that a unique function of Pol4 is to actively stabilize template strands that lack stacking continuity. We also explored whether NHEJ end processing can occur via short- and long-patch pathways analogous to base excision repair. Results demonstrated that 5' dRPs could be removed in the absence of Pol4 lyase activity. The 5' flap endonuclease Rad27 was not required for repair in this or any situation tested, indicating that still other NHEJ 5' nucleases must exist.
Assuntos
Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Bases , Dano ao DNA , DNA Polimerase beta , DNA Fúngico/genética , DNA Polimerase Dirigida por DNA/química , Endonucleases Flap/metabolismo , Modelos Moleculares , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Nonhomologous end joining (NHEJ) in yeast depends on eight different proteins in at least three different functional complexes: Yku70-Yku80 (Ku), Dnl4-Lif1-Nej1 (DNA ligase IV), and Mre11-Rad50-Xrs2 (MRX). Interactions between these complexes at DNA double-strand breaks (DSBs) are poorly understood but critical for the completion of repair. We previously identified two such contacts that are redundantly required for NHEJ, one between Dnl4 and the C terminus of Yku80 and one between the forkhead-associated (FHA) domain of Xrs2 and the C terminus of Lif1. Here, we first show that mutation of the Yku80 C terminus did not impair Ku binding to DSBs, supporting specificity of the mutant defect to the ligase interaction. We next show that the Xrs2-Lif1 interaction depends on Xrs2 FHA residues (R32, S47, R48, and K75) analogous to those known in other proteins to contact phosphorylated threonines. Two potential target threonines in Lif1 (T417 and T387) were inferred by identifying regions similar to a site in the human Lif1 homolog, XRCC4, known to be bound by the FHA domain of polynucleotide kinase. Mutating these threonines, especially T417, abolished the Xrs2-Lif1 interaction and impaired NHEJ epistatically with Xrs2 FHA mutation. Combining mutations that selectively disable the Yku80-Dnl4 and Xrs2-Lif1 interactions abrogated both NHEJ and DNA ligase IV recruitment to a DSB. The collected results indicate that the Xrs-Lif1 and Yku80-Dnl4 interactions are important for formation of a productive ligase-DSB intermediate.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
The ends of spontaneously occurring double-strand breaks (DSBs) may contain various lengths of single-stranded DNA, blocking lesions, and gaps and flaps generated by end annealing. To investigate the processing of such structures, we developed an assay in which annealed oligonucleotides are ligated onto the ends of a linearized plasmid which is then transformed into Saccharomyces cerevisiae. Reconstitution of a marker occurs only when the oligonucleotides are incorporated and repair is in frame, permitting rapid analysis of complex DSB ends. Here, we created DSBs with compatible overhangs of various lengths and asked which pathways are required for their precise repair. Three mechanisms of rejoining were observed, regardless of overhang polarity: nonhomologous end joining (NHEJ), a Rad52-dependent single-strand annealing-like pathway, and a third mechanism independent of the first two mechanisms. DSBs with overhangs of less than 4 bases were mainly repaired by NHEJ. Repair became less dependent on NHEJ when the overhangs were longer or had a higher GC content. Repair of overhangs greater than 8 nucleotides was as much as 150-fold more efficient, impaired 10-fold by rad52 mutation, and highly accurate. Reducing the microhomology extent between long overhangs reduced their repair dramatically, to less than NHEJ of comparable short overhangs. These data support a model in which annealing energy is a primary determinant of the rejoining efficiency and mechanism.
Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/metabolismo , Saccharomyces cerevisiae/genética , Oligonucleotídeos/genética , Proteína Rad52 de Recombinação e Reparo de DNA , Proteínas de Saccharomyces cerevisiaeRESUMO
The nonhomologous end-joining (NHEJ) pathway of DNA double-strand break repair requires three protein complexes in Saccharomyces cerevisiae: MRX (Mre11-Rad50-Xrs2), Ku (Ku70-Ku80), and DNA ligase IV (Dnl4-Lif1-Nej1). Much is known about the interactions that mediate the formation of each complex, but little is known about how they act together during repair. A comprehensive yeast two-hybrid screen of the NHEJ factors of S. cerevisiae revealed all known interactions within the MRX, Ku, and DNA ligase IV complexes, as well as three additional, weaker interactions between Yku80-Dnl4, Xrs2-Lif1, and Mre11-Yku80. Individual and combined deletions of the Yku80 C terminus and the Xrs2 forkhead-associated (FHA) domain were designed based on the latter two-hybrid results. These deletions synergistically blocked NHEJ but not the telomere and recombination functions of Ku and MRX, confirming that these protein regions are functionally important specifically for NHEJ. Further mutational analysis of Yku80 identified a putative C-terminal amphipathic alpha-helix that is both required for its NHEJ function and strikingly similar to a DNA-dependent protein kinase interaction motif in human Ku80. These results identify a novel role in yeast NHEJ for the poorly characterized Ku80 C-terminal and Xrs2 FHA domains, and they suggest that redundant binding of DNA ligase IV facilitates completion of this DNA repair event.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Antígenos Nucleares/metabolismo , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , Análise Mutacional de DNA , Reparo do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Autoantígeno Ku , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Telômero/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
In homologous recombination (HR), DNA polymerase δ-mediated DNA synthesis occurs within the displacement loop (D-loop) that is made by the recombinase Rad51 in conjunction with accessory factors. We describe in this chapter the reconstitution of the D-loop and repair DNA synthesis reactions using purified Saccharomyces cerevisiae HR (Rad51, RPA, and Rad54) and DNA replication (PCNA, RFC, and DNA polymerase δ) proteins and document the role of the Pif1 helicase in DNA synthesis via a migrating DNA bubble intermediate. These reconstituted systems are particularly valuable for understanding the conserved mechanism of repair DNA synthesis dependent on DNA polymerase δ and its cognate helicase in eukaryotic organisms.
Assuntos
Reparo do DNA , DNA/biossíntese , Recombinação Genética , Eletroforese em Gel de Poliacrilamida/métodosRESUMO
The glomerulus exercises its filtration barrier function by establishing a complex filtration apparatus consisting of podocyte foot processes, glomerular basement membrane and endothelial cells. Disruption of any component of the glomerular filtration barrier leads to glomerular dysfunction, frequently manifested as proteinuria. Ultrastructural studies of the glomerulus by transmission electron microscopy (TEM) and conventional scanning electron microscopy (SEM) have been routinely used to identify and classify various glomerular diseases. Here we report the application of newly developed helium ion scanning microscopy (HIM) to examine the glomerulopathy in a Col4a3 mutant/Alport syndrome mouse model. Our study revealed unprecedented details of glomerular abnormalities in Col4a3 mutants including distorted podocyte cell bodies and disorganized primary processes. Strikingly, we observed abundant filamentous microprojections arising from podocyte cell bodies and processes, and presence of unique bridging processes that connect the primary processes and foot processes in Alport mice. Furthermore, we detected an altered glomerular endothelium with disrupted sub-endothelial integrity. More importantly, we were able to clearly visualize the complex, three-dimensional podocyte and endothelial interface by HIM. Our study demonstrates that HIM provides nanometer resolution to uncover and rediscover critical ultrastructural characteristics of the glomerulopathy in Col4a3 mutant mice.